The present invention relates to modified human papillomavirus vectors and to methods of making and using the same, as non-integrating episomal plasmids and to certain DNA sequences, inserted therein or expressed within a host cell of the viral vector. In particular, the invention relates to a papillomavirus, in particular human papillomavirus, in which a naturally occurring genome of the virus has been altered (xe2x80x9cHPV vectorxe2x80x9d); to methods of making a vector and transducing cells therewith; to assure persistence of a vector, as an autonomous, extrachromosomal replicon maintained by the viral E1 and E2 replication proteins, despite the removal of all viral immortalization and transformation genes; and to certain DNA sequences inserted into or expressed in a host cell by way of an HPV vector, for purposes of long term gene transduction and expression for applications in genetic modification and therapy.
Since the development of recombinant DNA technology some 25 years ago, the prospect of developing extra-chromosomal gene expression vectors capable of long-term expression in transduced cells and/or tissues has become a reality with this invention. Prior developments in gene therapy research utilized vectors which either integrated the vector genome into the host chromosomes or were replication defective and non-persistent. The integration of the vector genome into a host chromosome is a potential and real cause of chromosomal insertional mutations and is vulnerable to unpredictable or uncontrollable inactivation of a reporter gene or a replacement gene. Retroviral vectors and adenovirus associated virus (AAV) vectors are representative examples of such insertional vectors. Alternatively, replication defective vectors may provide for cytoplasmic expression of vector genetic material within a host cell, but at the expense of the transduction behavior being lost over time in proliferating cells. Defective adenovirus and virus vectors are illustrative of vectors capable of transient expression in eukaryotic host cells, but with transduction being lost in proliferating cells. Other widely recognized deficiencies of certain viral vectors are their immunogenicity, and complications due to (a) prior immunity leading to immediate rejection of the infected cells and (b) induction of acute immune or hyperimmune responses within the first or second exposures during delivery of the vector to a host organism.
Thus, a novel vector capable of episomal replication transgene and regulated transcription can facilitate persistence and expression of a transgene in host cells without the limitations associated with insertional mutagenesis or disregulation or transgene expression disruption due to integration. These limitations have been observed in many in vivo studies using adenovirus or AAV vectors. J. M. Wilson, New Eng. J. Med. 1996, 334: 185-187. The human papillomaviruses have several attractive properties that are useful in augmenting gene expression and extrachromosomal maintenance in transfected cells.
The large family of papillomaviruses are significant human pathogens that infect epithelial tissues at selected body sites. Upon primary infection, or subsequent immunosuppression, papillomaviruses typically induce overt lesions variously called warts, papillomas or condylomata. These may go into short or long term remission. Nevertheless, viral DNA characteristically persists as episomes in stem cells. The episomal viral DNA may reactivate upon immunosuppression. A. Ferenczy et al., New Eng. J. Med. 1985; 313: 784-788, and A. Maran, Virology 1995; 212: 285-294. The latent phase of HPV infections is readily detected by Polymerase Chain Reaction (PCR) amplification of DNA. High levels of viral DNA amplification and mRNA transcription typically require terminal differentiation of squamous epithelia, and encapsidation into daughter viruses occurs in the superficial keratinocytes, prior to keratinocytes sloughing off the surface of the lesion. L. T. Chow and T. R. Broker, Viral Pathogenesis, N. Nathanson (Ed.), Lippincott-Raven, 1996, pp. 276-302. Well over 30 genotypes of human papillomavirus have an affinity for mucosal epithelia. E. M. de Villiers, Clinics in Dermatology 1997 (G. Orth and S. Jablonska, eds); 15: 199-206. Among the mucoscotrophic human papillomavirus strains are HPV-6, HPV-11, and HPV-16. While infections with HPV-16 occasionally progress to high-grade lesions and carcinomas, infections by HPV-6 or HPV-11 are almost universally benign and rarely reach such an advanced level of progression. In fact, most humans are infected with one or more types of cutaneous or mucosal HPV during their lifetime, and the virus persists thereafter under a degree of immunological surveillance and control, but without complete elimination. In almost all cases, such persistent maintenance remains subclinical with a small possibility of overt sporadic viral expression. For this and other reasons, HPVs are attractive vectors for long term gene therapy.
The HPV genome is a closed circular double-stranded DNA molecule, typically 7.9 kilo base pairs (kb) long. All HPV genotypes have a similar genome organization. The genomic organization of HPVs is known to the art and a representative prototype thereof, HPV-11, is set forth in FIG. 1. HPV genomes replicate as multi-copy nuclear, extrachromosomal plasmids. The differences in pathogenesis among various strains of papillomavirus mainly reside in the viral encoded E6 and E7 genes and in the upstream regulatory region (URR).
The lesions and possible carcinomas associated with papillomavirus infections are not associated with expression of HPV E1 and E2 replication proteins; rather the severe pathogenesis is initiated by inopportune expression of the viral oncogenes E6 and E7. The high risk oncogenic HPV-16, HPV-18 and related virus strains can cause disregulation in cell growth, cell differentiation and apoptosis. The natural functions of the oncoproteins are to reactivate the host DNA replication machinery in order to facilitate viral DNA amplification in differentiated keratinocytes that otherwise no longer express the host replication proteins. Consistent with these functions, HPV E6 and E7 genes are normally under the control of a differentiation-dependent upstream regulatory region (URR) promoter.
The E7 protein interacts with and inactivates the retinoblastoma susceptibility protein, pRB, a host tumor suppressor protein. M. Scheffner, et al. Curr. Top. Microbiol Immunology, 1994; 186: 83-99. This and/or related interactions reactivate the entire host DNA replication machinery in differentiated cells so that the virus DNA can amplify. Host DNA also replicates under these conditions as a by-product of the action of E7. S. Cheng, et al. Genes Dev. 1995; 9:2335-2349. The E6 protein inactivates another tumor suppressor protein, p53, thereby allegedly inhibiting premature apoptosis mediated by p53 when unscheduled DNA replication occurs in the differentiated cells in response to HPV E7 function. The cumulative result of HPV E6 and E7 protein activity in the differentiated stem cells can, in combination with presently unknown mutations in cellular genes, result in the immortalization of human keratinocytes in vitro or in host lesions. These proteins also are capable of transforming primary rodent epithelial cells in concert with an activated oncogene such as c-ras or c-fos. L. T. Chow and T. R. Broker, Ibid; and M Scheffner, et al., Ibid.
Downstream of the E2 gene in an HPV genome is the E5 gene, as shown in FIG. 1. HPV E5 protein is not an oncogene; however, it enhances the signal transduction of the EGF (epidermal growth factor) receptor, and can cause other membrane alterations. H. Stoppler, et al. Intervirology 1994; 37:168-179. Thus, HPV E5 protein augments the immortalization function of E6 and E7, yet E5 alone is not capable of immortalizing primary cells. M. C. Stoppler, et al., Virology, 1996; 223:251-254. While the role of HPV E5 is not fully appreciated in the literature, the bovine papillomavirus type 1 (BPV-1) E5 is the major viral oncogene. A key distinction from the HPVs, BPV-1 E5 functions to transform established rodent cell lines in vitro. BPV-1 E5 is detected in basal as well as in suprabasal differentiated cells of bovine warts caused by BPV-1. In light of the pathogenic nature of BPV E5 protein as well as other BPV proteins, such vectors raise a concern about the potential of E5-like proteins for oncogenesis. Previous attempts to design a viral vector suitable for episomal expression of foreign DNA in eukaryotes have met with only limited success due partly to the inability to separate deleterious viral genes from essential viral regulatory sequences. The instant invention largely overcomes these difficulties concerning vector design because the HPV oncoproteins are distinct from the HPV proteins necessary to support DNA replication from the native viral origin sequences. L. T. Chow and T. R. Broker, Ibid. The E6 and E7 viral oncogenes are normally up-regulated upon differentiation in HPV infected cells.
Similarly, HPV E4 protein function is not fully understood. E4 is known to associate directly or indirectly with cytokeratin intermediate filaments, but additional functions are postulated to exist. It is known, however, that HPV E4 is not an oncogene.
In contrast, to other members of the papovavirus family, such as mouse polyomavirus, simian virus 40, and human BK and JC viruses, for which the viral replication initiator and the oncogenic proteins are one and the same, HPV DNA replication utilizes host-encoded DNA replication enzymes, illustratively including DNA polymerases xcex1 and xcex4, PCNA, RF-C, RPA, DNA ligases and topoisomerases I and II. In addition, HPV replication utilizes host enzymes for the synthesis of deoxyribonucleoside triphosphate substrates. The virus contributes three components that are highly conserved among the papillomaviridae, which include: (1) the origin sequence (ori), (2) the ori recognition protein E2; and (3) the initiator protein E1. L. T. Chow and T. R. Broker, Interviroloy 1994, 37: 150-158; and A. Stenlund, DNA Replication in Eukaryotic Cells 1996: pp. 679-697.
The ori consists of binding sites (BS) for the two viral proteins E1 and E2 and is located within a noncoding, transcription regulatory region of approximately 750-1000 base pairs, designated the upstream (5xe2x80x2) regulatory region (URR). The URR also contains numerous host transcription factor binding sites that are thought to confer tissue tropism. H. U. Bernard and D. Apt, Arch. Dermatol. 1994, 130:210-215. E2 protein binds tightly to the multiple E2BS in the URR and helps recruit E1 to the origin. In addition, upon binding to E2BS, E2 also functions as a transcription factor and activates a surrogate promoter at a distance, depending on its concentration and those of other, host transcription factors. Hirochika et al., Genes Dev. 1988, 2:54-67; McBride et al., J. Biol. Chem. 1991, 225:18411-18414; Ham et al., Trends in Biochemical Sciences 1991, 16:440444. In its capacity as a replication and transcription factor, E2 bound to E2BS prevents nucleosome formation around the origin and help recruit E1 and perhaps host proteins to the origin to establish an initiation complex. A complex of approximately 6 copies of E1 which dimerizes to form a double hexamer is a helicase and helps unwind the ori and recruit the host DNA polymerase a to initiate replication. The E1 complex is required continuously during the elongation phase of replication, unlike the transient requirement for E2 during assembly of the pre-initiation complex on the origin. Because of sequence and functional conservation, E1 and E2 proteins encoded by the same virus can promote the replication of either a homologous or a heterologous papillomaviral origin. C. M. Chiang et al., Proc. Natl. Acad. Sci. USA 1992, 89:5799-5803; A. Del Vecchio et al., J. Virol. 1992, 66:5949-5958; F. Sverdrup and S. A. Khan, J. Virol. 1994, 68:505-509. Furthermore, replication from an HPV origin is not restricted to human or epithelial cells as long as adequate levels of E1 and E2 proteins are produced/generated from expression vectors. C. M. Chiang et al., Ibid. This is the basis for transient replication in transfected culture cells. M. Ustav and A. Stenlund, EMBO, J. 1991, 10:449-457. From their biochemical properties, it is clear that the E1 and E2 viral replication proteins are not oncoproteins.
The current knowledge of the mechanisms of HPV pathogenesis and of origin-specific DNA replication are utilized in the instant invention to design HPV based vectors without the risk of introducing viral oncogenes into a host cell. Thus, the instant invention exploits the property of HPV that the viral replication components and viral oncogenes are separable. The most commonly used strategies for delivering potential therapeutic genes into cells are based on retroviruses, adenoviruses, adenovirus-associated (parvo)virus (AAV), and recombinant DNA plasmids. Each has its limitations. Moreover, expression from the retroviral LTR tends to be down-regulated. The use of cellular gene promoters may overcome the problem. For adenoviruses, the strong host immune reaction to the highly immunogenic capsid proteins may prevent repeated application. AAV is limited by its size to approximately 5 kb of inserted DNA, and it integrates into host chromosomes nonspecifically. Transfection of plasmid DNA into cells is relatively inefficient compared to virus infection. Considerable efforts have been devoted to improve the efficiency of DNA transfection. A. R. Thierry et al., Proc. Natl. Acad. Sci. USA 1996, 193:11454-11459; E. R. Lee et al., Human Gene Therapy 1996, 7:1701-1717; N. Oudrhiri et al., Proc. Natl. Acad. Sci. USA 1997, 94:1651-1656. But the effectiveness of plasmid DNA is additionally restricted by the short duration in transfected cells for lack of replication of the transfected DNA. Papillomaviruses have properties that circumvent many of these impediments.
The instant invention is based upon the modification of the naturally occurring papillomavirus genome to produce vectors by rearrangement of the natural genome, by the removal of DNA from the genome, and by the introduction into the naturally occurring HPV genome of foreign DNA. Foreign DNA is defined herein as DNA naturally occurring in an organism other than HPV, or of synthetic origin. The genetic information designed for expression from the vector is introduced into a host eukaryote via a trophic vector. A vector of the instant invention represents an innocuous eukaryotic cloning vector for the expression of foreign DNA. The proclivity of HPV to infect specific eukaryotic epithelial cell types, the ability to separate the replication mediating viral gene sequences from those sequences responsible for oncogenesis and cellular growth control, and the capability to regulate the expression of the viral replication genes and transgenes by using previously characterized host or viral enhancers and promoters form the basis of the instant invention.
There exists a need for a gene therapy method that overcomes the problems of low-level transgene expression and expression for only a limited duration. The present invention fulfills this long-standing need in the art.
Vectors and methods are provided for introducing genetic material into host mammalian cells. More particularly, vectors and methods are provided for transferring a transgene to mammalian epithelial cells by way of a vector derived from a papillomavirus, such that the transgene undergoes episomal maintenance and expression. The instant invention harnesses the selectivity of a papillomavirus for specific mammalian host cell types, but is not restricted to epithelial cells. The episomal replication characteristics of HPVs are utilized for episomal transgene expression. In preparing a vector of the instant invention, gene sequences of the HPV genome which are oncogenic which otherwise up-regulate cell growth and/or DNA replication are excised in the course of preparing the vector.
A vector of the instant invention functions to deliver a transgene for episomal gene expression in a host cell. A vector of the instant invention generically contains: a transgene having an open reading frame or other desired genetic sequence and under the control of a surrogate promoter, and gene sequences expressing the HPV or other papillomavirus viral replication initiator proteins E1 and E2, expression of these proteins being controlled by another surrogate promoter, wherein the transgene and viral replication protein gene sequences are either contained within a single plasmid or located on two separate plasmids, which are cotransfected as an operative vector. The expression of the transgene, E1 and E2 all benefit by the presence of HPV URR. For the purposes of modification to achieve transfection in diverse cell types, the expression of the E1 and E2 and transgenes is or can be regulated by other enhancer-promoter units.
Upon transfection, the vector of the instant invention persists as an autonomous replicon following cell division an/or differentiation. The transgene is selected to serve any number of utilities, illustratively including: correction of an autosomal or somatic genetic or metabolic deficiency; expression of a viral or prokaryotic protein or immunogenic fragment thereof, expression of a polypeptide foreign to the host; and modulation of: hyperproliferative diseases (such as cancers), apoptotic diseases, and states of infectious diseases of viral, bacterial, or protozoan origin for purposes of control, reversal, cure or vaccination.